Optimising Electrical Interfacing between the Trimeric Copper Nitrite Reductase and Carbon Nanotubes.

The immobilization of copper-containing nitrite reductase (NiR) from Alcaligenes faecalis on functionalised multi-walled carbon nanotube (MWCNT) electrodes is reported. It is demonstrated that this immobilization is mainly driven by hydrophobic interactions, promoted by the modification of MWCNTs with adamantyl groups. Direct electrochemistry shows high bioelectrochemical reduction of nitrite at the redox potential of NiR with high current density of 1.41 mA cm-2 . Furthermore, the desymmetrization of the trimer upon immobilization induces an independent electrocatalytic behavior for each of the three enzyme subunits, corroborated by an electron-tunneling distance dependence.

A Pyrene-Triazacyclononane Anchor Affords High Operational Stability for CO2 RR by a CNT-Supported Histidine-Tagged CODH.

An original 1-acetato-4-(1-pyrenyl)-1,4,7-triazacyclononane (AcPyTACN) was synthesized for the immobilization of a His-tagged recombinant CODH from Rhodospirillum rubrum (RrCODH) on carbon-nanotube electrodes. The strong binding of the enzyme at the Ni-AcPyTACN complex affords a high current density of 4.9 mA cm-2 towards electroenzymatic CO2 reduction and a high stability of more than 6×106  TON when integrated on a gas-diffusion bioelectrode.

Inhibition of Urease, a Ni-Enzyme: The Reactivity of a Key Thiol With Mono- and Di-Substituted Catechols Elucidated by Kinetic, Structural, and Theoretical Studies.

The inhibition of urease from Sporosarcina pasteurii (SPU) and Canavalia ensiformis (jack bean, JBU) by a class of six aromatic poly-hydroxylated molecules, namely mono- and dimethyl-substituted catechols, was investigated on the basis of the inhibitory efficiency of the catechol scaffold. The aim was to probe the key step of a mechanism proposed for the inhibition of SPU by catechol, namely the sulfanyl radical attack on the aromatic ring, as well as to obtain critical information on the effect of substituents of the catechol aromatic ring on the inhibition efficacy of its derivatives. The crystal structures of all six SPU-inhibitors complexes, determined at high resolution, as well as kinetic data obtained on JBU and theoretical studies of the reaction mechanism using quantum mechanical calculations, revealed the occurrence of an irreversible inactivation of urease by means of a radical-based autocatalytic multistep mechanism, and indicate that, among all tested catechols, the mono-substituted 3-methyl-catechol is the most efficient inhibitor for urease.

Insights into Urease Inhibition by N-( n-Butyl) Phosphoric Triamide through an Integrated Structural and Kinetic Approach.

The nickel-dependent enzyme urease represents a negative element for the efficiency of soil nitrogen fertilization as well as a virulence factor for a large number of pathogenic and antibiotic-resistant bacteria. The development of ever more efficient urease inhibitors demands knowledge of their modes of action at the molecular level. N-( n-Butyl)-phosphoric triamide (NBPTO) is the oxo-derivative of N-( n-butyl)-thiophosphoric triamide (NBPT), which is extensively employed in agriculture to increase the efficiency of urea-based fertilizers. The 1.45 Šresolution structure of the enzyme-inhibitor complex obtained upon incubation of Sporosarcina pasteurii urease (SPU) with NBPTO shows the presence of diamido phosphoric acid (DAP), generated upon enzymatic hydrolysis of NBPTO with the release of n-butyl amine. DAP is bound in a tridentate binding mode to the two Ni(II) ions in the active site of urease via two O atoms and an amide NH2 group, whereas the second amide group of DAP points away from the metal center into the active-site channel. The mobile flap modulating the size of the active-site cavity is found in a disordered closed-open conformation. A kinetic characterization of the NBPTO-based inhibition of both bacterial (SPU) and plant ( Canavalia ensiformis or jack bean, JBU) ureases, carried out by calorimetric measurements, indicates the occurrence of a reversible slow-inhibition mode of action. The latter is characterized by a very small value of the equilibrium dissociation constant of the urease-DAP complex caused, in turn, by the large rate constant for the formation of the enzyme-inhibitor complex. The much greater capability of NBPTO to inhibit urease, as compared with that of NBPT, is thus not caused by the presence of a P═O moiety versus a P═S moiety, as previously suggested, but rather by the readiness of NBPTO to react with urease without the need to convert one of the P-NH2 amide moieties to its P-OH acid derivative, as in the case of NBPT. The latter process is indeed characterized by a very small equilibrium constant that reduces drastically the concentration of the active form of the inhibitor in the case of NBPT. This indicates that high-efficiency phosphoramide-based urease inhibitors must have at least one O atom bound to the central P atom in order for the molecule to efficiently and rapidly bind to the dinickel center of the enzyme.

Urease Inhibition in the Presence of N-(n-Butyl)thiophosphoric Triamide, a Suicide Substrate: Structure and Kinetics.

The nickel-dependent enzyme urease is a virulence factor for a large number of pathogenic and antibiotic-resistant bacteria, as well as a negative factor for the efficiency of soil nitrogen fertilization for crop production. The use of urease inhibitors to offset these effects requires knowledge, at a molecular level, of their mode of action. The 1.28 Å resolution structure of the enzyme-inhibitor complex obtained upon incubation of Sporosarcina pasteurii urease with N-(n-butyl)thiophosphoric triamide (NBPT), a molecule largely utilized in agriculture, reveals the presence of the monoamidothiophosphoric acid (MATP) moiety, obtained upon enzymatic hydrolysis of the diamide derivative of NBPT (NBPD) to yield n-butyl amine. MATP is bound to the two Ni(II) ions in the active site of urease using a µ2-bridging O atom and terminally bound O and NH2 groups, with the S atom of the thiophosphoric amide pointing away from the metal center. The mobile flap modulating the size of the active site cavity is found in the closed conformation. Docking calculations suggest that the interaction between urease in the open flap conformation and NBPD involves a role for the conserved αArg339 in capturing and orienting the inhibitor prior to flap closure. Calorimetric and spectrophotometric determinations of the kinetic parameters of this inhibition indicate the occurrence of a reversible slow inhibition mode of action, characterized, for both bacterial and plant ureases, by a very small value of the dissociation constant of the urease-MATP complex. No need to convert NBPT to its oxo derivative NBPTO, as previously proposed, is necessary for urease inhibition.